Team:UNITN-Trento/Notebook
From 2013.igem.org
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</html>|pSpac-PXyl-GFP}} | </html>|pSpac-PXyl-GFP}} | ||
+ | |||
+ | == 90 == | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/Labpost|2013-10-07|gabriele-viola|Reboot: starting from the beginning|<html> | ||
+ | |||
+ | Since the new forward primer (with the entire prefix) arrived, I started again from the beginning.<br/> | ||
+ | <br/> | ||
+ | <h3>SAM synthetase extraction</h3> | ||
+ | The new primer forward has just the EcoRI restriction site added at its beginning (complete prefix):<br/> | ||
+ | GAATTCGCGGCCGCTTCTAGAGAAGGAGGAACTACTATGGCAAAACACCTTTTT<br/> | ||
+ | It has a Tm = 68.7°C.<br/> | ||
+ | <br/> | ||
+ | Then I performed two Phusion PCR (one GC and one HF) and one Phusion/RBC PCR to identify the best protocol for SAM synthetase extraction with the new primer. | ||
+ | |||
+ | </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Phusion PCRs|<html><center> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th colspan="3">PCR Mixes</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="border:none;"></td> | ||
+ | <th>Mix HF (1)</th> | ||
+ | <th>Mix GC (2)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Phusion Buffer HF</td> | ||
+ | <td>10µl</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Phusion Buffer GC</td> | ||
+ | <td>0</td> | ||
+ | <td>10µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dNTPs</td> | ||
+ | <td colspan="2">1µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>template</td> | ||
+ | <td colspan="2">1µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Primer Fw</td> | ||
+ | <td colspan="2" rowspan="2">2.5µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Primer Rv</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Phusion pol</td> | ||
+ | <td colspan="2">0.5µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Water</td> | ||
+ | <td colspan="2">33.5µl</td> | ||
+ | </tr> | ||
+ | </table><br/> | ||
+ | <br/> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th colspan="4">Phusion Settings</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Step</th> | ||
+ | <th>Temperature</th> | ||
+ | <th>Time</th> | ||
+ | <th>Go to</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>98°C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>98°C</td> | ||
+ | <td>10 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>72°C</td> | ||
+ | <td>35 sec</td> | ||
+ | <td>step #2, 30 times</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>72°C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>4°C</td> | ||
+ | <td>pause</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center></html>}}<html> | ||
+ | |||
+ | </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Phusion/RBC PCR|<html> | ||
+ | |||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th colspan="2">PCR Mix</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="border:none;"></td> | ||
+ | <td>Mix RBC (3)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Template</td> | ||
+ | <td>1µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dNTPs</td> | ||
+ | <td>0.5µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Primer Fw</td> | ||
+ | <td rowspan="2">1µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Primer Rv</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer RBC</td> | ||
+ | <td>5µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Phusion pol</td> | ||
+ | <td>0.3µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>RBC</td> | ||
+ | <td>0.25µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Water</td> | ||
+ | <td>40.95µl</td> | ||
+ | </tr> | ||
+ | </table><br/> | ||
+ | <br/> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th colspan="4">PCR Settings</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="border:none;"></td> | ||
+ | <th>Step</th> | ||
+ | <th>Temperature</th> | ||
+ | <th>Time</th> | ||
+ | <th>Go to</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>94°C</td> | ||
+ | <td>2 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>94°C</td> | ||
+ | <td>1 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>62.5°C</td> | ||
+ | <td>1 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>72°C</td> | ||
+ | <td>1 min 9 sec</td> | ||
+ | <td>step #2, 30 times</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>72°C</td> | ||
+ | <td><7 min/td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>4°C</td> | ||
+ | <td>pause</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html>}}<html> | ||
+ | <br/> | ||
+ | The products of these three PCRs were then loaded on a 1% agarose gel. | ||
+ | </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th colspan="4">Loading scheme</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1kb ladder</td> | ||
+ | <td>GC(2)</td> | ||
+ | <td>HF(1)</td> | ||
+ | <td>RBC(3)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/8/82/Tn-20130710-GG_SAMsynthetase_newPrimers_chosePCR.jpg" alt="Gel" width="450px"> | ||
+ | </html>}}<html> | ||
+ | As showed by the gel, only the Phusion/RBC PCR was successful.<br/> | ||
+ | <br/> | ||
+ | <hr> | ||
+ | <h3>Purifications</h3> | ||
+ | Then, I purified the EX-SAMsynthetase-SP sample produced today with the Phusion/RBC PCR, and the 5 R0010 PCR insert that were amplified on <a href="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-02-gabriele">tuesday 02/07</a>. | ||
+ | <center> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Type</th> | ||
+ | <th>Quantity</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>RBC(3)</td> | ||
+ | <td>EX-SAMsynthetase-SP</td> | ||
+ | <td>103.6ng/µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>HF1</td> | ||
+ | <td>R0010 insert</td> | ||
+ | <td>17.5ng/µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>HF2</td> | ||
+ | <td>R0010 insert</td> | ||
+ | <td>19.5ng/µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>HF3</td> | ||
+ | <td>R0010 insert</td> | ||
+ | <td>15.9ng/µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>G1</td> | ||
+ | <td>R0010 insert</td> | ||
+ | <td>17.8ng/µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>G2</td> | ||
+ | <td>R0010 insert</td> | ||
+ | <td>16.3ng/µl</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
+ | <br> | ||
+ | <hr> | ||
+ | <h3>OverNight Purification</h3> | ||
+ | Then, Viola was so polite to prepare the O/N digestion mixes (<a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion">with the usual protocol</a>) and incubate them at 37°C. An EX-SAMsynthetase-SP sample (RBC#3 from today, 103.6ng/µl) and a linear pSB1C3 sample (G3A from <a href="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele">01/07</a>, 44.8ng/µl) were restricted with XbaI and PstI (Nebuffer2). | ||
+ | <html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html><center> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <td style="border:none;"></td> | ||
+ | <th>RBC#3</th> | ||
+ | <th>G3A</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Template</td> | ||
+ | <td>40µl</td> | ||
+ | <td>50µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>XbaI</td> | ||
+ | <td rowspan="2">2.5µl</td> | ||
+ | <td rowspan="2">1.5µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PstI</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>NEBuffer 2</td> | ||
+ | <td>10µl</td> | ||
+ | <td>5µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BSA</td> | ||
+ | <td>10µl</td> | ||
+ | <td>5µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Water</td> | ||
+ | <td>35µl</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center></html>}}</html> | ||
+ | </html>|SAMsynthetase}} |
Revision as of 07:26, 22 July 2013
Home | Team | Official Team Profile | Project | Parts Submitted to the Registry | Modeling | Notebook | Safety | Attributions |
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You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.
1
We extracted BBa_J45319, BBa_J45320, BBa_J45004 and BBa_J45119 and then transformed them into NEB10β cells.
2
3
For the protocol used see the RBC Taq PCR protocol.. When the reaction finished, we tested the presence of the aplificate product througt an electrophoresis analisys (adding 2 µl of LD for 10 µl of DNA).
Results:
As you can see from our gel image, our product is present in both reactions (TAQ and TAQ+Phusion). After purifying our products we find out that the concentration of DNA that we obtained is too low (about 5ng/ul) maybe for an experimental error in setting the PCR program. For this reason, our team is going to redo the PCR reaction again.
4
Foward: GCCGCTTCTAGAGAAGGAGGAACTACTATGGCAAAACACCTTTTT Reverse: CTGCAGCGGCCGCTACTAGTATTATTACTTCAGACCGGCAG
For the protocol used see the Phusion PCR protocol.. When the reaction finished, we tested the presence of the aplificate product througt an electrophoresis analisys (adding 2 µl of LD for 10 µl of DNA).
Results:
As you can see from our gel image, our product is not present. The next move will be to try to amplify using a TAQ polymerase and we hope that this will work!
5
As you can see from the picture we both obtained great results
6
7
8
Sample | SAM Synthetase | pSB1C3 |
---|---|---|
1 | 18,6 ng/ul | 21,6 ng/ul |
2 | 16,2 ng/ul | 16 ng/ul |
That's definetely not a good result, however we will continue working with them.
9
10
11
part | plasmid | plate | colonies |
---|---|---|---|
Bba_J45119 | pSB1AT3 | amp, tet 10µg/ml, tet 50µg/ml | only in amp plate |
Bba_J45320 | pSB1AT3 | amp, tet 10µg/ml, tet 50µg/ml | only in amp and tet 10µg/ml |
Bba_J45 700 | pSB1AK3 | kan | yes |
12
We took the three inocula of SAMsynthetase+pSB1C3 and we performed the purification (Wizard Plus SV Miniprep DNA purification system). Then to verify if the colonies that we took contain the correct insert we perform the screening protocol and the gel eletrophoresis.
13
We digested using XbaI and PstI and purified the products previously obtained via PCR and via extraction from the registry (pSB1C3 with RFP). We then quantified them at the nano-drop.
pSB1C3 + RfP | 27,8 ng/ul |
---|---|
pSB1C3 linearized | 17,7 ng/ul |
SAM Synthetase | 13,2 ng/ul |
14
- K323002
- K143012
- K323000
- K323003
Results: since we didn't obtain any colonies we failed the experiment.
15
Inoculum of three B.subtilis backbone
We have done the inoculum of three Bacillus-specific backbone (previously transformed in NEB and plated on Ampicillin LB Agar) in 19 50ml Falcon - 7 with the part BBa_K823023 - 6 with the part BBa_K823022 - 6 with the part BBa_K823027 The Falcons were put in the incubator at 37 °C o/n.Digestion of SAMsynthetase and psB1C3
We have digested the SAMsynthetase gene (previously amplificated and purificated)and the linerarized vector psB1C3 with the enzyme Xba1 and Pst1 exploiting the same protocol. Then we have incubated the mix at 37C o/n.16
17
N.B.Some sample have shown an unexpected red color. There is the possibilities that some backbones contain the RFP, maybe all.
We have quantified the products with the following results:
BBa_K823023(4 sample):220 ng/μl , 228.9 ng/μl ,246.4 ng/μl , 272.3 ng/μl
BBa_K823022(3 sample): 222.8 ng/μl , 227.5 ng/μl ,284.9 ng/μl
BBa_K823027(2 sample):268 ng/μl , 299.3 ng/μl
Then we have tried to verify the parts digesting with EcoR1 and Pst1 and doing an electrophoresis, unfortunately it seems that we didn't load the marker Fermentas 1 KB.
18
Well | Loaded samples | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
1 | DNA Ladder 1Kb | |||||||||
2 | #1BBa_J823022(A) | |||||||||
3 | #2BBa_J823023(A) | |||||||||
4 | #4BBa_J823027(A) | |||||||||
5 | #5BBa_J823022(B) | |||||||||
6 | #6BBa_J823023(B) | |||||||||
7 | #7Bba_J823027(B) |
19
1) pSB1C3 with RFP (Ctrl_1)
2) pSB1C3 with RFP + SAMsynthetase (1:2)
3) pSB1C3 without RFP (Ctrl_2)
4) pSB1C3 without RFP (1:2)
The samples were prepared and ligation was performed following the ligation protocol.
- Plasmid concentration: 27.8ng/µl
- Plasmid length: 2070bp
- Insert concentration: 13.2ng/µl
- Insert length: 1155bp
- Used plasmid: 250µl
- Volume of reaction: 35µl
- Buffer concentration: 10X
Transformation in NEB10β cells was performed following the transformation protocol. Plates in incubator ON at 37°C static, more than 16 hours.
20
Particular care to use Distilled water for Ampicillin and Ethanol for Chloramphenicol.
21
We have prepared plate at the concentration of 50 um/ml for ampicillin and 150 ul/ml for chloramphenicol.
Seems that the concentration of Chloramphenicol is 5 times more concentrated than normally used.
22
Since we received the Ethylen Forming Enzyme gene from Genescript company, we proceeded with the extraction and the transformation test. We resuspended the 4 ug of DNA in 40 ul of sterilized water (obtaining a 100 ng/ul stock solution). After that, we transformed 200 ul of NEB10 competent cells with 1ul of EFE DNA. Finally we plated them in two 50 ug/ml Amp LB-Agar Petri dishes.
Results:
As you can see from the image, we obtained many colonies. We then inculated x5 colonies in 5ml LB with Ampicillin.
23
Me and Bruno transformed the parts that LMU Munich sent us in NEB10beta cells line. The following transformation worked:
Bba_K823022 (pSBBS4S)
Bba_K823027 (pSBBS2E)
Bba_K823023 (pSBBS1C)
24
We followed the protocol for the transformation efficiency kit to determine how efficiency are our competent cells. We performed 5 transformations with 5 different concetrations of the part Bba_J04450 in the plasmid pSB1C3.
25
- SAMsynthetase+RFP (1:2) = too many to count.
- SAMsynthetase+RFP (ctrl) = too many to count.
- SAMsynthetase (1:2) = 11 colonies.
- SAMsynthetase (ctrl) = 12 colonies.
Performed 5 inocula for each of the two 1:2 plates, in 5ml LB with 5µl CM. Tomorrow miniprep, quantification, digestion and gel to control what happened.
26
- 4 out of 5 inocula of SAMsynthetase+pSB1C3[no_RFP] were good, one (3) didn't grow.
- 4 out of 5 inocula of SAMsynthetase+pSB1C3[RFP] were good, one (5R) was red!
Then, we miniprepped the samples (except for the red, 5R, one) using the protocol. We chose to miniprep also the inocula that did not grow (3), just as a control. After miniprep, quantification was performed with nanodrop.
Sample | DNA [ng/µl] | Sample | DNA [ng/µl] |
---|---|---|---|
1 | 173.7 | 1R | 157.0 |
2 | 188.2 | 2R | 97.1 |
3 | 18.6 | 3R | 148.0 |
4 | 178.7 | 4R | 143.7 |
5 | 103.3 | 5R | - |
After that, we digested an aliquote of 800ng of each DNA sample with EcoRI and PstI using our digestion protocol, putting the digestion mix to incubate for 1.5h at 37°C (static).
1 | 2 | 4 | 5 | 1R | 2R | 3R | 4R | |
---|---|---|---|---|---|---|---|---|
Buffer 10X (Nebuffer4) | 2µl | |||||||
EcoRI | 1µl | |||||||
PstI | 1µl | |||||||
BSA 10X | 1µl | |||||||
Template | 4.6µl | 4.3µl | 4.3µl | 7.75µl | 5.1µl | 8.2µl | 5.4µl | 5.6µl |
Water (up to 20µl) | 10.4µl | 10.7µl | 10.7µl | 7.25µl | 9.9µl | 6.8µl | 9.6µl | 9.4µl |
Total volume | 20µl | |||||||
BamHI | 1µl |
Then we realized that LacI+RFP and SAMsynthetase have the same base length, and that a gel would not be able to discriminate between them. So, we added 1µl of BamHI to each sample, knowing that this enzyme is able to cut SAMsynthetase (in a band of 100bp and one of 1055bp) to identify the presence of SAM.
So, we prepared a 1.5% agarose gel and performed an electrophoresis for 30 minutes at 120V.
Loaded samples | |||||||||
1kb ladder | #1 | #2 | #4 | #5 | 100bp ladder | #1R | #2R | #3R | #4R |
To determine whether the insert was LacI+RFP or SAMsynthetase we wanted to perform a RBC Taq PCR against SAMsynthetase (SAMsynthetase-Fw and SAMsynthetase-Rv primers), but we set a wrong PCR program... next time verify the PCR program more accurately!!! On Monday we will start again from the PCR... so sad D:
27
28
29
Next I perfomed six different ligations:
two controls with: the pSB1C3 linearized and not but without the insert
two with: pSB1C3 linearized or not and BMST1
two with: pSB1C3 linearized or not and PCHA
30
31
Loading scheme | ||||
G1 | G2 | 1kb ladder | E1 | E2 |
Sadly, something went wrong with G2 sample (probably Gabriele forgot to add something to the PCR mix). But the gel is very beautiful!!!
Then G1, E1 and E2 samples were purified using Wizard® SV Gel and PCR Clean-Up System and then quantified using the Nanodrop.
Sample | Quantity |
---|---|
G1 | 80.2ng/µl |
E1 | 65.7ng/µl |
E2 | 60ng/µl |
Finally we prepared overnight digestion of SAMsynthetase (G1 and E1, E2 was put at -20°C) and pSB1C3 linearized both with XbaI and PstI-HF, using the digestion protocol. We used Nebuffer4 and the mix were prepared as follows:
G1 | E1 | linear pSB1C3 | |
---|---|---|---|
Nebuffer4 | 10µl | 5µl | |
XbaI | 1µl | ||
PstI-HF | 1µl | ||
BSA | 1µl [from 100X stock] | 5µl [from 10X stock] | |
DNA [3µg] | 37.41µl | 45.66µl | 37.31µl |
Water | 49.59µl | 41.34µl | 0.69µl |
Total | 100µl | 50µl |
32
33
During these 1.5 hours, we performed the miniprep (protocol) and quantification of the circular pSB1C3 inocula of yesterday.
Sample | Quantity |
---|---|
G1 | 184.1 ng/µl |
G2 | 187.7 ng/µl |
G3 | 165.7 ng/µl |
E1 | 117.3 ng/µl |
E2 | 105.7 ng/µl |
E3 | 99.0 ng/µl |
After the incubation, we purified the digestion mixes using the Wizard® SV Gel and PCR Clean-Up System and then quantified.
Sample | Quantity |
---|---|
linear pSB1C3 | 30.0 ng/µl |
SAMsynthetase G1 | 40.4 ng/µl |
SAMsynthetase E1 | 32.8 ng/µl |
SAMsynthetase E1 was stocked at -20°C.
Then, we performed the ligation of pSB1C3 and SAMsynthetase exploiting the usual protocol.
Ctrl | 1:1 | 1:2 | 1:4 | |
---|---|---|---|---|
Buffer | 2.5µl | 3.0µl | ||
Plasmid | 10 µl | |||
Insert | 0 | 4.14µl | 8.28µl | 16.56µl |
Ligase | 2µl | |||
Water | 10.5µl | 6.36µl | 2.22µl | 0 |
Total | 25µl |
Also, we extracted R0010 promoter (Plac) from the registry (2013 distribution kit, plate 3, well 3H). Unfortunately, we also extracted a part from the same plate and well of the 2012 distribution kit (BBa_K115032) because we mistook the kits.
Finally, we transformed NEB10β competent cells with the usual protocol, we used the following quantities of DNA: 10µl of each ligation product except for the 1:4 ligation, of which we used 15µl, and 1µl of the extracted R0010. Then, we plated on CM plates.
34
I began the week doing some minipreps (x5 of EFE and x5 of pSB1C3+RFP) following this protocol.
Sample | EFE | pSB1C3 |
---|---|---|
1 | 253,8 ng/ul | 161,9 ng/ul |
1 | 243,5 ng/ul | 160,9 ng/ul |
3 | 261,4 ng/ul | 142,5 ng/ul |
4 | 218,0 ng/ul | 168,4 ng/ul |
5 | 299,1 ng/ul | sample lost |
I then digested the linearized pSB1C3 (500 ng, kindly offered by Caterina), pSB1C3 + RFP (500 ng) and EFE (1000 ng) with EcorI and PstI following the 2A assembly protocol. After that, I proceeded with the ligation and transformation in NEB10B cells.
35
I inoculated the previously transformed EFE in pSB1C3 and AraCpBAD into 5ml of LB containing Chloramphenicol.
36
37
38
39
40
EFE in pSB1C3 | ng/ul | AraCpBAD | ng/ul |
---|---|---|---|
1:4 n°1 | 552,1 | 1 | 285,0 |
1:4 n°2 | 482,0 | 2 | 311,5 |
1:4 n°3 | 558,9 | 3 | 446,8 |
1:2 n°1 | 779,6 | 4 | 404,8 |
1:2 n°2 | 376,1 | 5 | 442,6 |
1:1 n°1 | 359,6 | ||
1:1 n°2 | 501,6 |
41
Then we prepared a gel (1% GellyPhor gel) and checked for the products.
Loaded samples | Result |
---|---|
1kb ladder | - |
empty | - |
B1 | OK |
B2 | OK |
B3 | OK |
E1 | NO |
E2 | NO |
E3 | NO |
G1 | NO |
G2 | OK |
G3 | OK |
empty | - |
1kb ladder | - |
Then we (GG, ET) have proceeded with the inoculation of pSB1C3+SAMsynthetase (1:1, 1:2, 1:4) and of part R0010 (pLac). Unfortunately there were a few colonies also in the control.
The inocula were performed in plastic culture tubes (15ml) instead of the glass ones, because we do not know how to sterilize the latter. The tubes were filled with 5ml of LB with Cloramphenycol (1µl of 34mg/ml Cloramphenycol stock solution for 1ml of LB).
In the afternoon Gabriele performed again the same PCR protocol to linearize pSB1C3. He loaded the E1-3 samples again, as double-check.
Loaded samples | Result |
---|---|
G1A | NO |
G2A | OK |
G3A | OK |
G4A | OK |
1kb ladder | - |
E1 | NO |
E2 | NO |
E3 | NO |
42
For the digested vector (AraCpBAD), we incubated the part for one hour at 37°C with the SAP phosphatase before disactivating the enzymes.
We then preceeded by ligating and transforming the two parts in competent NEB10b cells following the ligation protocol for Biobricks .
We decided to do that in duplicate and with different ratios plasmid:insert (ctrl, 1:1, 1:2, 1:4) obtaining so 8 reactions.
Results: as you can see from the pictures, we there are only few colonies in the control so we hope our construct is correctly cloned. The next step will be the inocula and the screening test.
43
I inoculated the previously transformed AraCpBAD + EFE in pSB1C3 into 5ml of LB containing Chloramphenicol.
As you can see, it seems that all the inocula grown. I will proceed with the purification and the screening test!
44
Sample | ng/ul |
---|---|
1:1 Plate1 n°1 | 1201,5 |
1:1 Plate1 n°2 | 1620,5 |
1:2 Plate1 n°1 | 969,8 |
1:4 Plate1 n°1 | 636,3 |
1:1 Plate2 n°1 | 672,8 |
1:1 Plate2 n°2 | 1222,5 |
1:2 Plate2 n°1 | 782,0 |
1:4 Plate2 n°1 | 705,3 |
As you can see, 6 samples out of 8 have the expected bands: 2070bp for the vector and 2300bp for AraCpBAD.
45
46
Sample | ng/ul |
---|---|
K090501 | 117,1 |
K090504_a | 115,3 |
K090504_b | 101,3 |
K823000_a | 156,0 |
K823000_b | 171,1 |
K823000 | 115,5 |
I used a 1.5% concentration of agarose to create a gel and I used Ethidium bromide instead the normal EuroSafe. In addition I have not used a normal Dye with the 20ul of sample loaded but I used 4ul of 30% glycerol.
49
50
Sample | Quantification (ng/µl) |
---|---|
1:1 A | 293.0 |
1:1 B | 135.7 |
1:1 C | 178.0 |
1:1 D | 139.3 |
1:1 E | 262.0 |
1:1 F | 147.5 |
1:1 G | 212.5 |
1:1 H | 210.2 |
1:2 A | 141.0 |
1:2 B | 123.6 |
1:4 A | 207.8 |
1:4 B | 127.2 |
R0010 A | 131.7 |
R0010 B | 119.6 |
R0010 C | 371.0 |
R0010 D | 307.9 |
R0010 E | 313.8 |
Loading Scheme |
---|
Empty |
1kb ladder |
Empty |
1:1 D |
1:1 E |
Empty |
1:2 B |
Empty |
1:4 A |
Empty |
R0010 E |
Loading scheme |
---|
1kb ladder |
R0010 A |
R0010 B |
R0010 C |
R0010 D |
R0010 E |
Loading scheme |
---|
R0010 B |
R0010 C |
R0010 D |
1kb ladder |
1:4 A |
1:4 B |
1:2 A |
1:2 B |
Loading scheme |
---|
1kb ladder |
R0010 A |
R0010 B |
R0010 C |
R0010 D |
R0010 E |
51
Sample | Quantities |
---|---|
A | 198 ng/µl |
B | 187.6 ng/µl |
C | 138 ng/µl |
E | 179.9 ng/µl |
F | 200.2 ng/µl |
G | 197.2 ng/µl |
After that, I have digested 800ng of each sample with EcoR1-HF and Pst1-HF and one (F) also with Xba1 and Pst1-HF following the digestion protocol after 30min at 37°C I have run the final pruduct on a gel with no results again:
I have re-re-tried to linearize pSB1C3 (in triplicates) with no results (in the second line we can see some aspecific bands): Exploiting the iGem 2012 kit I have transformed 2µl of the pLac promoter (R0010 in pSB1A2) in NEB10β competent cells following the transformation protocol. Afterwards, I plated them on an ampicillin-containing plate.
52
The LB medium contained Ampicillin (1:1000).
53
Sample | Quantity |
---|---|
1 | 396.1ng/µl |
2 | 523.1ng/µl |
3 | 171ng/µl |
Quantity | |
---|---|
Water | 4µl |
Sharpmass Euroclone 100bp ladder | 1µl |
glicerol solution(30%) | 1µl |
Sample | Well |
---|---|
2 µl loading dye | 1 |
Ladder | 2 |
sample 1 | 3 |
sample 2 | 4 |
sample 3 | 5 |
sample 4(Bruno e Fabio) | 6 |
sample 4(Bruno e Fabio) | 6 |
Ladder 1000 kb | 7 |
loading dye | 8 |
We can see 2 bands: the highest is the backbone (pSB1A2: 2079bp) and the lower is pLac (nearly 243bp, located between the bands 300 and 200bp of the ladder).
After the screening I digested 3µg of the pSB1A2+R0010 (sample #2) with SpeI and PstI-HF o/n following the usual protocol.
54
We performed the digestion and the ligation on:
1a)BBa_K1065101 with EcoR I and Xba I and 1b)BBa_J45320 with EcoR I and Spt I
to obtain the complete device BBa_K1065102(placI+PchBA+araC-pBAD promoter+BMST1 in pSB1C3)
2a)pSB1C3 linearized with EcoR I and Pst I 2b)BBa_J45320 with EcoR I and Pst I
to obtain the device BBa_K1065103 (placI+PchBA in pSB1C3).
At the end of the day we did the trasformaion in NEB10β cells. Waiting for the results...break a leg!
55
Ctrl | 1:1 | 1:3 | |
---|---|---|---|
Buffer | 2.5µl | ||
plasmid | 5.73µl | ||
insert | 0 | 3.09µl | 9.27µl |
ligase | 1µl | ||
water | 15.77µl | 12.68µl | 6.50µl |
Gabriele prepared also 4 inocula from the pSB1C3+R0010 CM-plate, just to be sure that it is (not) there.
56
I have tried to amplify the sample of 117.3 ng/µl, 105.7 ng/µl and one of Girelli's sample(184.1 ng/µl)following the Phusion PCR protocol and the universal primers.
No one of this PCRs has succeded!
57
ID | Quantification | RBS strength |
---|---|---|
BBa_E0240 | 108.5 ng/µl | medium |
BBa_E0840 | 129 ng/µl | strong |
Moreover Emil did the inocula (in LB + Ampicillin) of two backbones(BBa_823024 and BBa_823026) from the plates of Fabio and Bruno(2 inocula for each sample).
58
In order to evalutate if the expression of EFE is toxic or not to our cells I created a growth curve. I started from an inoculum of AraCpBAD + EFE that I prepared yesterday. I diluited 50ul of the overnight culture in 5ml of LB and I incubated at 37C with shaker until the cultures reached approximately 0,6 O.D at 600nm. I took 2 samples for negative controls and 2 samples for the induced cells. After that I added 25ul of Arabinose 1M stock solution to the induced samples (5mM working solution) and I registered the O.D. of the samples one time per hour (for 4 hours). In the end I plotted the results.
As expected, the strong induction of our gene slightly influences on growth-rate (caused by stress) but still is not completely toxic.
To confirm this result I made a toxicity test by serial dilution. I diluited 50ul of overnight culture (one induced sample and one not induced) into 9,5ml of LB. After vortexed the samples, I further diluited them 1:10 for three times. In the end I plated 150ul of each dilution onto Chloramhenicol Plates.
As you can see from the images, there's not big differences in the colony forming ability of the induced and the not induced sample, even in a dilution of 1:1000. Maybe I should dilute them more!
59
The results of the transformations were not so good. We've only few colonies on each plate (only on Michele's control there were many colonies!). Despite this, we've decided to do inocula of some colonies in the afternoon. On the rest of the day Michele tried two different PCRs to obtain pSB1C3 linearized (someone stole his supplies!!). Obviously no results (immagini gel?). Caterina repeated the digstion but she used two differents type of ligase for the reaction of ligation. At the end of the day she transformed Nebα with these new products of ligation.
60
Sample | Quantification |
---|---|
BBa_K823024 | 334.4ng/µl |
BBa_K823024 | 260.6ng/µl |
BBa_K823026 | 306.7ng/µl |
BBa_K823026 | 359.7ng/µl |
61
Sample | Quantification |
---|---|
BBa_E0840/1 | 484.6ng/µl |
BBa_E0840/2 | 376ng/µl |
BBa_E0840/3 | 481.8ng/µl |
BBa_E0840/4 | 475ng/µl |
BBa_E0240/1 | 361ng/µl |
BBa_E0240/2 | 454.4ng/µl |
BBa_E0240/3 | 368ng/µl |
BBa_E0240/4 | 365.8ng/µl |
Sample | Well |
---|---|
Ladder 1kb Fermentas | 1 |
BBa_E0840/1 | 2 |
BBa_E0840/3 | 3 |
BBa_E0240/2 | 4 |
BBa_E0240/4 | 5 |
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Sample | Quantity (ng/µl) |
---|---|
#A | 243.7 |
#B | 194.1 |
#C | 245.3 |
#D | 252.9 |
Then, the four quantified samples were screened after a digestion with ExoRI-HF and PstI-HF, with the usual screening protocol (incubated only for 45min, since both the enzymes are HF).
The digestion products were then run on a gel with a transparent loading dye (30% glycerol): each loaded sample contained 16µl of the sample and 4µl of transparent loading dye. Both a 1kb normal ladder and a 100bp transparent ladder were loaded, also the first well was loaded with the usual loading dye.
Loading scheme |
---|
Loading dye |
Empty |
100bp ladder |
#A |
#B |
#C |
#D |
Empty |
Empty |
1kb ladder |
Finally, Emil prepared the inocula of the product of ligation of R0010 and SAMsynthetase (in pSB1A2: 6 inocula of the 1:1 ligation product and 3 of the 1:3 ligation producta).
63
Sample | Well |
---|---|
BBa_E0840/2 | 2 |
BBa_E0840/4 | 3 |
BBa_E0240/1 | 4 |
BBa_E0240/3 | 5 |
Ladder 1kb Fermentas | 1 |
Sample | Well |
---|---|
BBa_E0840/4 a | 2 |
BBa_E0840/4 b | 3 |
BBa_E0840/4 c | 4 |
Ladder 1kb Fermentas | 1 |
Sample | Quantification |
---|---|
BBa_E0840/4 a | 30.5ng/µl |
BBa_E0840/4 b | 44ng/µl |
BBa_E0840/4 c | 42.6ng/µl |
64
Sample | Quantification |
---|---|
BBa_E0840 | 15.8ng/µl |
BBa_K823026 | 37.6ng/µl |
BBa_K823024 | 36.4ng/µl |
Then I performed the ligation(1:1,1:3,CTRL) of the GFP with the 2 backbone individually and following the ligation protocol.Finally I transformed 10 µl of the ligation protocol in Neb10β and plate them on ampicillin LB agar following the transformation protocol.
65
•
Sample | ng/ul |
---|---|
K823001 a | 436.8 |
b | 275.4 |
c | 466.5 |
K823002a | 138.0 |
b | 134.4 |
c | 139.7 |
K823003 a | 85.4 |
b | 99.9 |
c | 118.9 |
K143012 a | 178.3 |
b | 89.2 |
c | 121.4 |
K823024 a | 362.1 |
d | 209.3 |
e | 288.2 |
K823026 a | 246.3 |
b | 315.8 |
c | 550.3 |
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68
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69
•
70
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Nutrient agar (100 ml) : 0,8 g nutrient broth: 1,5g Agar; water to 100 ml.
Nutrient agar + starch (100 ml) : 0,8 g nutrient broth: 1,5g Agar; 2ml starch from potato, water up to 100 ml.
Nutrient broth (250 ml): 2 g nutrient broth; 250 ml water.
Nutrient broth + starch (250 ml): 2 g nutrient broth; 5 ml starch; water up to 250 ml;
To propagate di original bacillus pellet, we resuspended it in 1 ml of nutrient broth+starch and put this milliliter in 5 ml of nutrient broth+starch for a final 6 ml mother liquid culture. Then we made several other liquid cultures with different bacteria concentrations from the Mother (for each concentration we made both starch and non-starch liquid cultures). We put all in a shaker at 26 degrees. We plated also the same concentrations in several plates with both Nutrient agar + starch and Nutrient agar alone, and put them at 26. Just out of curiosity we decided to put some plates and some liquid cultures at 37 degrees.
71
From the survivor we made further liquid cultures ( at 26°, 1:50 and 1:100; at 37° 1:50 and 1:100)
As soon as they became very cloudy we went along with the glycerol stock preparation and put our bacillus at -80°. The one that weren’t cloudy have been kept in the shaker all night long.
In the afternoon we inoculated some colonies from the plates in 5 ml of nutrient broth +starch.
72
73
Starting from an overnight culture, I diluted it 1:100 and I incubated it until it reached 0,5 O.D.600. After that, I added 5mM of Arabinose and I incubated the culture at 37°C for about 4-5 hours. In the I connected the vial previously keeped ermetically closed at the micro GC and I took the measure. I did this work for three samples: negative control (not induced), 5mM Arabinose V=1,5ml and 5mM Arabinose V=3ml.
Sample | Ethylene detected |
---|---|
Not induced | 0±15 ppm |
5mM Arabinose V=1,5ml | 30±15 ppm |
5mM Arabinose V=3ml | 70±15 ppm |
74
Today was a nice day! Finally Michele’s plate were positive (no colonies on the control and lot of them on the others!) So at the end of the day we could do the inocula. Moreover Michele finished the reaction of ligation with the plasmid pSB1C3 linearized (digested the day previous by Caterina (link post)) and J45319 and transformed it in 200 ul of NebB10. At the end of the day Caterina plate them. Everything is working. Hoping that Mesa will be detectable!
75
You know, after a BBBQ, everything is more difficult!! In particular today was an exausting day. While in the morning Caterina did three miniprep from the inocula (post del giorno prima metti link = 4 with araCpBAD + BSTM1 in pSB1C3 and 1 only control of Neb10&beta, mettere immagine del gel di cate) Michele took the two inocula that left and did 7 diluition 1:100 in 20 mL of LB. In particular he did 2 diluition for the control and five for the bacteria with the plasmid. He put them at 37°C in agitation waiting for them reaching O.D. = 0.5. After four hours he did the induction with arabinosio even if the bacteria with the plasmid were at about 0.3 O.D. The induction was done only on five samples to have another control. After two hours it was added different concentrations of Salycilic Acid (in solution with H20 and etanol) in different samples and after two more hours we did a SNIFF Test to understand if some Mesa was produced. The results were not so brilliant: in fact the bacteria in which were added higher concentrations of SA stinked a bit less and were more fresh. Durante la giornata cate ha fatto anche pcr per J45119 ma fail (immagine) In più fatte prime misure con GC per vedere detabilità mesa (grafici)
76
77
Sample | Quantification |
---|---|
K823026+E0840 1:3 A | 409.4ng/µl |
K823026+E0840 1:3 B | 401.0ng/µl |
K823026+E0840 1:1 C | 733.4ng/µl |
K823026+E0840 1:1 D | 489.3ng/µl |
K823024+E0840 1:1 E(succesful) | 281ng/µl |
K823024+E0840 1:3 F | 341.1ng/µl |
K823024+E0840 1:3 G | 220.3ng/µl |
K823024+E0840 1:1 error | 129.3ng/µl |
Sample | Well |
---|---|
Ladder 1kb Fermentas | 1 |
BBa_K823026+BBa_E0840 A | 2 |
BBa_K823026+BBa_E0840 B | 3 |
BBa_K823026+BBa_E0840 C | 4 |
BBa_K823026+BBa_E0840 D | 5 |
BBa_K823024+BBa_E0840 E(the only succesful) | 6 |
BBa_K823024+BBa_E0840 F | 7 |
BBa_K823024+BBa_E0840 G | 9 |
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Sample | Quantification |
---|---|
1:1 024+0840 E | 482.6ng/µl |
1:1 024+0840 E | 700ng/µl |
1:1 024+0840 | 373.1ng/µl |
1:3 024+0840 | 423.5ng/µl |
1:3 024+0840 | 327.8ng/µl |
1:1 026+0840 | 293.1ng/µl |
1:1 026+0840 | 1243ng/µl |
1:3 026+0840 | 766.4ng/µl |
1:3 026+0840 | 369.1ng/µl |
GFP | 15.9ng/µl |
K823026 | 36.3ng/µl |
K823026 | 43.7ng/µl |
Sample | Well |
---|---|
Ladder 1kb Fermentas | 1 |
BBa_K823024+BBa_E0840 A | 3 |
BBa_K823024+BBa_E0840 B | 5 |
BBa_K823024+BBa_E0840 C | 6 |
BBa_K823024+BBa_E0840 D | 7 |
BBa_K823024+BBa_E0840 E | 8 |
BBa_K823026+BBa_E0840 F | 9 |
BBa_K823026+BBa_E0840 G | 10 |
BBa_K823026+BBa_E0840 H | 11 |
BBa_K823026+BBa_E0840 I | 12 |
N.B. Remember always to spin the ligation buffer before using
After that I plated the product of the ligation as done before(1:1,1:3,ctrl for each sample) on Ampicillin added LB.
79
Sample | Quantities |
---|---|
BBa_K823026+BBa_E0840(1:1) 1 | 438.8 ng/µl |
BBa_K823026+BBa_E0840(1:1) 2 | 400.7 ng/µl |
BBa_K823024+BBa_E0840(1:1) 3 | 343.6 ng/µl(the only succesful) |
Sample | Well |
---|---|
Ladder 1kb Fermentas | 1 |
BBa_K823026+BBa_E0840(1:1) a | 3 |
BBa_K823026+BBa_E0840(1:1) b | 4 |
BBa_K823024+BBa_E0840(1:1) | 5 |
As we can see only the third(024) sample shows the insert at the right size of 1000 bp(the lower band), then I did the inocula of the plates of 5/07(1:1,1:2,1:3) and of the old plate that gives right results to amplify 024(1:1).
80
The idea is to grow into a beaker our strain of E. coli that produces ethylene, and connect it to a laboratory dryer with bananas. In this way, E. coli (be stirred and 37 ° C), producing Ethylene, should increase the maturation of the banana in the dryer connected.
In addition, we have included in another beaker Mesa pure with LB, which should slow down the ripening at high concentration and should promote the ripening at low concentrations.
81
Sample | Quantities |
---|---|
BBa_K823024+BBa_E0840(1:1) 2 | 267 ng/µl |
BBa_K823024+BBa_E0840(1:1) 3 | 248.6 ng/µl |
Sample | Well |
---|---|
BBa_K823024+BBa_E0840(1:1) b | 1 |
BBa_K823024+BBa_E0840(1:1) | 2 |
Ladder 1kb Fermentas | 3 |
82
(1) PCR: SAM synthetase amplification
I amplified SAM synthetase by performing a PCR on the samples G1 and E2 from 17/06.Sample | Quantification |
---|---|
G1 | 80ng/µl |
E2 | 60ng/µl |
The PCR was performed following the usual SAM extraction protocol.
G1 mix | E2 mix | |
---|---|---|
Template(50ng) | 0.63µl | 0.83µl |
dNTPs | 0.5µl | |
primer Fw | 1µl | |
primer Rv | 1µl | |
buffer RBC | 5µl | |
Phusion pol | 0.3µl | |
RBC pol | 0.25µl | |
water | 41.32µl | 41.12µl |
PCR results were then run on a 1% agarose gel:
Loading scheme | ||
---|---|---|
1kb ladder | G1 | E2 |
Since the gel shows two bands at nearly 1200bp, the PCR results are confirmed. The PCR products were then purified with the Promega kit.
Sample | Type | Quantity |
---|---|---|
G1 | SAM synthetase | 35.6ng/µl |
E2 | SAM synthetase | 31.5ng/µl |
(2) pSB1A2+R0010+SAMsynthetase ligation screening
Then I screened the inocula from the 26/06 ligation. First I miniprepped the inocula:1:1 | A | 490.9ng/µl |
B | 252.1ng/µl | |
C | 345.6ng/µl | |
D | 295.3ng/µl | |
E | 315.6ng/µl | |
F | 226.2ng/µl | |
1:3 | A | 345.7ng/µl |
B | 147.0ng/µl | |
C | 268.0ng/µl |
The samples were then digested using the screening digestion protocol and then run on a 1% agarose gel.
Loading scheme |
---|
1kb ladder |
1:1 A |
1:1 B |
1:1 C |
1:1 D |
1:1 E |
1:1 F |
1kb ladder |
1:3 A |
1:3 B |
1:3 C |
Given that the gel shows only two bands (one at nearly 2kbp and the other at 200bp), SAM synthetase is not present and the ligation failed.
(3) Linear pSB1C3 purification
I also purified with the usual protocol the linearized pSB1C3 from 19/06.Sample | Type | Quantity | |
---|---|---|---|
G2 | linear pSB1C3 | 74.4ng/µl | |
G3 | linear pSB1C3 | 63.3ng/µl | |
G2A | linear pSB1C3 | 44.0ng/µl | |
G3A | linear pSB1C3 | 44.8ng/µl | |
G4A | linear pSB1C3 | 41.9ng/µl |
(4) O/N Digestion
Finally, I prepared an overnight digestion of the samples G1 (SAM synthetase) and G2 (linear pSB1C3) to try again the ligation tomorrow. I followed the usual protocol.G1 SAMsynthetase | G2 linear pSB1C3 | |
---|---|---|
template (3µg) | 48.5µl | 40µl |
XbaI | 2.5µl | 1.5µl |
PstI | 2.5µl | 1.5µl |
NEBuffer 2 | 10µl | 5µl |
water | 26.5µl | 0 |
83
SAM synthetase ligation in (linear) pSB1C3
First of all, early in the morning I added 1µl of DpnI to the SAMsynthetase#G2 O/N digestion and 1µl of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37°C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80°C).Then, I purified and quantified the two digestion samples:
Sample | Quantity |
---|---|
G1 SAMsynthetase | 18.8ng/µl |
G2 linear pSB1C3 | 6.5ng/µl |
After that, I performed the ligation (the plasmid had a too low concentration).
Ctrl | 0.5:1 | 0.5:2 | |
---|---|---|---|
Buffer | 3µl | ||
Plasmid | 14.3µl | ||
Insert | 0 | 6µl | 12µl |
Ligase | 1µl | ||
Water | 11.7µl | 5.7µl | 0µl |
And, "finally", I transformed the ligations in NEB10β cells.
(2) SAMsynthetase amplification
I amplified SAMsynthetase from the E2 sample (that was purified yesterday) following the usual protocol. The PCR was performed in triplicates.The PCR products were run on a 1% agarose gel.
Loading scheme | ||||
---|---|---|---|---|
E1A | E1B | E1C | empty | 1kb ladder |
As shown in the gel, a band at nearly 1200bp is present, confirming the success of the PCR.
(3) R0010 amplification
I also amplified R0010 from A sample (R0010, 243.7ng/µl) from 27/06. Being the first time amplifying this sequence, I performed (in triplicates or duplicates) a Phusion PCR using as primers the prefix Fw (Tm = 86°C) and the suffix Rv (Tm = 90°C).Mix HF (x3) | Mix GC (x2) | |
---|---|---|
Phusion GC buffer | 0 | 10µl |
Phusion HF buffer | 10µl | 0 |
dNTPs | 1µl | |
primer Fw | 2.5µl | |
primer Rv | 2.5µl | |
template (50ng/µl) | 0.5µl | |
Phusion pol | 0.5µl | |
Water | 33µl |
Given that R0010 is 200bp long, the PCR program was the following:
PCR setting | |||
---|---|---|---|
Step | Temperature | Time | Go to |
1 | 98°C | 30 sec | |
2 | 98°C | 10 sec | |
3 | 72°C | 3 sec | step #2, 30 times |
4 | 72°C | 10 min | |
5 | 4°C | pause |
Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.
Loading scheme |
---|
100bp ladder |
AHF1 |
AHF2 |
AHF3 |
AGC1 |
AGC2 |
empty |
1kb ladder |
As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!
(4) linear pSB1C3 amplification
I didn't know that linear pSB1C3 is "impossible" to amplify, and that the only way to get it is to linearize the circular one. So I tried its amplification and failed.Loading scheme | |||
---|---|---|---|
1kb ladder | G3A | G3B | G3C |
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A sample of the same stock culture was kept in thermoshaker. As expected an higher value of ethylene was detected (see the green point) since it was subjected to only one measure (thus having no gas loss due to repeated measures).
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Interestingly, inducing with arabinose when the sample has reached OD600 0.8 causes an increase of more than two fold in the production of ethylene.
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Sample | Quantities |
---|---|
BBa_K823026+BBa_E0840(1:1) | 421.3ng/µl |
BBa_K823026+BBa_E0840(1:1) | 438.6ng/µl |
BBa_K823026+BBa_E0840(1:3) | 356.7ng/µl |
BBa_K823026+BBa_E0840(1:3) | 415.2ng/µl |
BBa_K823024+BBa_E0840 | 401.2ng/µl |
BBa_K823024+BBa_E0840 | 365ng/µl |
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SAM synthetase extraction
The new primer forward has just the EcoRI restriction site added at its beginning (complete prefix):GAATTCGCGGCCGCTTCTAGAGAAGGAGGAACTACTATGGCAAAACACCTTTTT
It has a Tm = 68.7°C.
Then I performed two Phusion PCR (one GC and one HF) and one Phusion/RBC PCR to identify the best protocol for SAM synthetase extraction with the new primer.
PCR Mixes | ||
---|---|---|
Mix HF (1) | Mix GC (2) | |
Phusion Buffer HF | 10µl | 0 |
Phusion Buffer GC | 0 | 10µl |
dNTPs | 1µl | |
template | 1µl | |
Primer Fw | 2.5µl | |
Primer Rv | ||
Phusion pol | 0.5µl | |
Water | 33.5µl |
Phusion Settings | |||
---|---|---|---|
Step | Temperature | Time | Go to |
1 | 98°C | 30 sec | |
2 | 98°C | 10 sec | |
3 | 72°C | 35 sec | step #2, 30 times |
4 | 72°C | 10 min | |
5 | 4°C | pause |
PCR Mix | |
---|---|
Mix RBC (3) | |
Template | 1µl |
dNTPs | 0.5µl |
Primer Fw | 1µl |
Primer Rv | |
Buffer RBC | 5µl |
Phusion pol | 0.3µl |
RBC | 0.25µl |
Water | 40.95µl |
PCR Settings | ||||
---|---|---|---|---|
Step | Temperature | Time | Go to | |
1 | 94°C | 2 min | ||
2 | 94°C | 1 min | ||
3 | 62.5°C | 1 min | ||
4 | 72°C | 1 min 9 sec | step #2, 30 times | |
5 | 72°C | <7 min/td> | ||
6 | 4°C | pause |
The products of these three PCRs were then loaded on a 1% agarose gel.
Loading scheme | |||
---|---|---|---|
1kb ladder | GC(2) | HF(1) | RBC(3) |
Purifications
Then, I purified the EX-SAMsynthetase-SP sample produced today with the Phusion/RBC PCR, and the 5 R0010 PCR insert that were amplified on tuesday 02/07.Sample | Type | Quantity |
---|---|---|
RBC(3) | EX-SAMsynthetase-SP | 103.6ng/µl |
HF1 | R0010 insert | 17.5ng/µl |
HF2 | R0010 insert | 19.5ng/µl |
HF3 | R0010 insert | 15.9ng/µl |
G1 | R0010 insert | 17.8ng/µl |
G2 | R0010 insert | 16.3ng/µl |
OverNight Purification
Then, Viola was so polite to prepare the O/N digestion mixes (with the usual protocol) and incubate them at 37°C. An EX-SAMsynthetase-SP sample (RBC#3 from today, 103.6ng/µl) and a linear pSB1C3 sample (G3A from 01/07, 44.8ng/µl) were restricted with XbaI and PstI (Nebuffer2). {{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|RBC#3 | G3A | |
---|---|---|
Template | 40µl | 50µl |
XbaI | 2.5µl | 1.5µl |
PstI | ||
NEBuffer 2 | 10µl | 5µl |
BSA | 10µl | 5µl |
Water | 35µl | 0 |